V5-76-005
PERSISTENCE AND DEGRADABILITY TESTING OF
BENZIDINE AND OTHER CARCINOGENIC COMPOUNDS
June 1976
FINAL REPORT
-------
This document is available to the public through the
National Technical Information Service
Springfield, Virginia 22151
-------
EPA 560/5-76-005 TR 76-571
PERSISTENCE AND DEGRADABILITY TESTING OF
BENZIDINE AND OTHER CARCINOGENIC COMPOUNDS
Authors
Philip H. Howard
Jitendra Saxena
Contract No. 68-01-2679 Task 2
June 1976
Project Officer
Carter Schuth
Prepared for
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
-------
This draft report has been reviewed by the Office
of Toxic Substances, EPA, and approved for publi-
cation. Approval does not signify that the con-
tents necessarily reflect the views and policies
of the Environmental Protection Agency, nor does
mention of trade names or commercial products
constitute endorsement or recommendation for use.
ii
-------
TABLE OF CONTENTS
Page
ABSTRACT v
I. Introduction 1
II. Review of the Analytical Method Used in SOCMA Studies 2
III. Summary and Evaluation of Technical Information Developed on 6
Environmental Degradation of Benzidine
A. Die-Away Tests in Chlorinated, Aerated, and 6
Non-Aerated Lake Water
B. Photolytic Studies in Deionized Aerated Water 9
C. Biological Degradation - Activated Sludge System 13
D. Benzidine Ambient Monitoring Data 15
E. Comparison of SOCMA Conclusions and Conclusions Supported 16
by Research Data
IV. General Discussion of Environmental Degradation Testing as 18
Applied to Aromatic Amines and Carcinogens
V. Conclusion ' 21
REFERENCES 22
iii
-------
LIST OF TABLES
Number Page;
1 Benzidine Degradation Under Various Lake Water Conditions 7
2 Benzidine Degradation in Chlorinated and Aerated Lake Water 8
3 Photolysis Study of 100 ppb Benzidine in Deionized, Aerated Water 11
4 Photolysis of Benzidine at Various Concentrations in Deionized, 12
Aerated Water Initially at 6.9 - 7.0 pH
iv
-------
ABSTRACT
This report reviews and evaluates information on the environmental de-
gradation of benzidine which was generated by the Synthetic Organic Chemical
Manufacturer's Association (SOCMA) Benzidine Task Force and submitted to the
U.S. Environmental Protection Agency. The experimental design, execution,
and interpretation of the studies have been reviewed and evaluated. It is
concluded that the SOCMA information is not sufficient for making intelligent
14
regulatory decisions, and it is recommended that a test program using C-
benzidine be undertaken in order to generate the necessary information.
-------
I. INTRODUCTION
The U.S. Environmental Protection Agency has been working on promulgating
the Toxic Pollutant Effluent Standard for benzidine under Section 307(a) of the
Federal Water Pollution Control Act Amendments of 1972. At the same time, the
Office of Toxic Substances has been developing its capability to implement the
concepts embodied in the Toxic Substances Control Act regarding the need for
increased environmental testing of industrial chemicals. Understanding the
fate of benzidine is, thus, important not only to the development of an effec-
tive effluent standard but also as an example of voluntary industrial testing
for environmental effects. Benzidine, besides being an important comnercial
chemical, is well-known to be carcinogenic: incidence of bladder tumors among
workers exposed to benzidine is high, and the compound is reported to induce
hepatic tumors in mice and intestinal tumors in rats and to accelerate the
appearance of breast cancer in female rats.
A review of the available literature (Radding et al., 1975) conducted for
the Office of Toxic Substances, EPA, revealed that very little was known about
the environmental fate of benzidine. In an effort to fill this information
gap, the Synthetic Organic Chemical Manufacturer's Association (SOCMA) Benzi-
dine Task Force has been working since 1973 to generate information necessary
for the development of the benzidine standard. The SOCMA Benzidine Task Force
has made three submissions to EPA which have included information on benzidine
degradation. The purpose of this report is to review the experimental design,
execution, and interpretation of these SOCMA submissions. EPA's interests in
the analysis of the information include: (1) regulatory decisions to be made
on benzidine; (2) the environmental fate of aromatic amines as a class; and
(3) the adequacy of the benzidine testing program as a prototype for environ-
mental fate testing. These interests will also be addressed in this report.
-------
II. REVIEW OF THE ANALYTICAL METHOD USED IN THE SOCMA STUDIES
Analytical methods used to study environmental degradation of a chemical
can have considerable impact on the interpretation of the results. In all the
SOCMA studies (SOCMA, 1975 a, b) a Chloramine-T colorimetric method was used.
The method has been proposed by EPA as an acceptable method for monitoring
benzidine and its salts in wastewater. When used with water samples, the method
consists of the following steps:
1. Make sample basic (benzidine hydrochloride converted to undissociated
benzidine).
2. Extract benzidine into ethyl acetate.
3. Extract benzidine from ethyl acetate with IN hydrochloric acid.
4. Add Chloramine-T (Sodium toluene-p-sulfonchloroamide) to oxidize
benzidine to a yellow meriquinoid complex.
H2N-
~Na++ HC1
HN =
•NH
H2N<5>
\__/
•/O^NH2
2HC1
5.
HN =( ) =\ > =NH
\—/ \—=»/
Meriquinoid Complex
Extract yellow meriquinoid complex with ethyl acetate (sometimes
chloroform) and record the absorption spectrum from 510 nm to 370 nm
(benzidine meriquinoid complex has a maximum absorbance at 436 nm)
for quantitative purposes.
-------
This method appears to be extremely sensitive and specific for benzidine
and its salts in water. The U.S. EPA (1974) stated that the detection limit
was 0.2 ]ig/H, (ppb) when analyzing 1 liter samples. Allied Chemical (Puliafico,
1975) has had some experience applying the method to wastewaters and they con-
cluded that the limit .of detection is not more than 0.5 ppb at 50% background
transmission with a 5 cm cell. When the background is greater than 50% (com-
monly the case with dye manufacturing plant waste), the limit of detection
may be greater than 5 ppb. When relatively uncontaminated water is used, a
low detection limit of 0.02 yg/fc (0.02 ppb) was reported by the Great Lakes
Laboratory (SOCMA, 1975 a, b).
Besides being sensitive, the Chloramine-T colorimetric method is also
fairly specific for benzidine. Any interferences would have to be extrac-
\
ted into ethyl acetate from basic solution, extracted back into hydrochloric
acid, and develop a colored product on treatment with Chloramine-T that could
be extracted back into ethyl acetate. Analogs of benzidine, such as dichloro-
benzidine, £-tolidine, and dianisidine (Classman and Meigs, 1951) can interfere,
but compounds such as 2,4-diaminobiphenyl, aniline, a- and g-naphthylamine
give no color on Chloramine-T treatment and, therefore, do not interfere (Butt
and Strafford, 1956). Recording the visible spectrum of the complex also adds
an extra degree of specificity, since the interfering analogs have different
maximum absorptions.
Thus, overall, the analytical method used in the SOCMA submissions is a
very sensitive and specific method for only benzidine or its salts. This con-
clusion in itself is an important one which impacts on the interpretation of
all of the results. Because of the selected analytical method, or any method
that measures only the parent compound, all the measurements of benzidine
-------
degradation are really only measurements of the loss of benzidine. Very slight
alterations may result in no detection by the analytical method used. For
example, Sciarini and Meigs (1958) noted that although the Chloramine-T method
is very sensitive for benzidine, it is "much less sensitive for 3-monohydroxy-
benzidine, 3,3'-dihydroxybenzidine, and £-aminophenol." In fact, dihydroxy-
benzidine added to urine could not be extracted with any solvent.
With benzidine, slight chemical changes might be quite significant. It
is postulated that carcinogenic aromatic amines must undergo metabolic activa-
tion to a proximate carcinogen prior to tumor induction. The following ring-
and N-hydroxylated products are regarded as the likely proximate carcinogens
of benzidine (Gadian, 1975; Arcos and Argus, 1975):
3-hydroxybenzidine
3,3'-dihydroxybenzidine
N-hydroxybenzidine
N, N'-dihydroxybenzidine
H2N-
-------
These compounds might be formed from benzidine, but would not be detected
by the Chloramine-T method. Thus, although the Chloramine-T method would measure
the loss of benzidine, that loss may not correspond to a total degradation of
its likely proximate carcinogens.
-------
III. SUMMARY AND EVALUATION OF TECHNICAL INFORMATION DEVELOPED ON ENVIRONMENTAL
DEGRADATION OF BENZIDINE
A. Die-Away Tests in Chlorinated, Aerated, and Non-Aerated Lake Water-
Experimental Design, Results, and Evaluation
Two experiments on the degradation of benzidine in lake water were
conducted. The first (SOCMA, 1975 a) consisted of measuring the loss of benzi-
dine in lake water (obtained from a Buffalo, NY pumping station) which was:
(1) maintained at 1 mg/SL available chlorine, (2) maintained at 2 mg/£ available
chlorine, (3) stirred, (4) aerated, and (5) undisturbed. Initial benzidine
concentrations of 1, 2, and 5 yg/£ were used and all colorimetric analyses
were performed in triplicate. The investigators indicated that all attempts
were made to shield the experimental set-up from light. The results are pre-
sented in Table 1.
In the second study on benzidine degradation in water, three condi-
tions were used: (1) lake water brought to an initial chlorine concentration
of 1 ppm, (2) lake water brought to an initial chlorine concentration of 2 ppm,
and (3) vigorously aerated lake water (approximately 10 H of air per hour).
The initial benzidine concentration ranged between 1 to 70 ppb. Benzidine and
dissolved oxygen levels were measured at various intervals. Table 2 summarizes
the results. The investigators subjected the data to statistical analysis and
concluded that the data showed no significant differences under the different
conditions. By plotting the reaction rates, the investigators concluded that
the reaction was probably 1st order with a rate constant of 0.175 per hour
(half-life = 4 hours).
These results indicate rather conclusively that something is happening
at a relatively fast rate to benzidine that is placed in lake water. However,
-------
Table 1. Benzidine Degradation Under Various Lake Water Conditions (SOCMA, 1975 a)
Benzldlne Concentration (pg/8.)
Conditions
Chlorinated Lake Water
(1 mg/e C12)
Chlorinated Lake Water
(2 mg/i C12)
Aerated Lake Water
Stirred Lake Water
Undisturbed
Lake Water
Time Solution A
Oirs.) (initial ^ 1 ue/)O
0.5
4
12
24
48
72
0.5
4
12
24
48
72
0.5
4
12
24
48
72
0.5
4
12
24
48
72
0.5
4
12
24
48
72
0.84
0.48
0.10
N.D.
-
—
0.79
0.39
0.04
N.D.
-
-
0.83
0.69
0.31
N.D.
-
—
0.90
0.78
0.48
N.D.
-
-
0.89
0.71
0.47
0.02
N.D.
—
Solution B
(Initial ^ 2 ufc/8.)
1.90
1.17
0.68
N.D.
.
-
1.73
1.09
0.31
N.D.
-'
-
l.')4
l.Ai.
0.7.J
N.J).
-
-
1.8:
l..")7
0.80
N.n.
-
-
1.85
1.45
0.76
N.D.
-
-
Solution C
(initial •»- 5 ue/O
4.42
4.06
2.17
N.D.
-
-
4.37
4.10
1.87
N.D.
-
-
4.61
4.27
2.01
N.D.
-
-
4.70
4.21
2.15
N.D.
-
-
4.8J
4.03
2.07
N.D.
-
-
N.D. •= Not detectable (<0.02 wg/4)
-------
Table 2. Benzidine Degradation in Chlorinated and Aerated Lake Water (SOCMA, 1975 b).
Benaidine Concentration
oo
Conditions
Chlorinated
Lake Water
1 ppm C12
Chlorinated
Lake Water
2 ppm C12
Aerated
Lake Water
Time
(hrs.)
0.5
3
6
12
24
0.5
3
6
12
24
0.5
3
6
12
24
Initial 1 *
Benzidine 0
0.89
0.51
0.39
0.09
<0.02
0.92
0.49
0.28
0.10
<0.02
0.87
0.58
0.40
0.28
<0.02
92.3
89.2
87.3
88.0
85.6
91.7
89.7
88.6
87.2
88.0
100
100
100
100
100
Initial 5 * Initial 10 A
Benzidine 0. Benzidine 0
4.47
3.97
2.93
2.03
<0.02
4.81
3.87
3.01
1.83
<0.02
91.6
87.6
87.3
88.2
86.3
92.3
87.8
86.9
85.4
86.7
9.70
6.81
5.37
4.18
0.73
9.64
6.95
5.08
3.73
0.52
9.37
8.02
5.43
4.00
0.13
93.0
89.3
88.1
87,9
88.0
96.4
88.3
88.0
87.3
86.9
100
100
100
100
100
Initial 50 *
Benzidine C<
47.36
36.21
28.34
18.69
1.05
46.73
34.77
29.32
17.97
1.21
94.7
87.4
88.3
86.9
85.8
94.4
89.1
88.3
86.9
87.7
Initial 70 ^
Benzidine 0
68.33
42.76
32.81
21.84
1.62
67.24
43.81
30.96
21.32
1.19
66.90
43.51
32.84
25.00
1.46
93.2
88.1
87.8
88.1
88.0
93.2
88.0
89.2
87.6
88.0
100
100
100
100
100
Dissolved oxygen (% saturation)
-------
as previously discussed, the analytical method allows no insight into the
degree or pathway of degradation. After 48 hours, no benzidine is detectable
by the colorimetric method, even at relatively high starting concentrations.
Unfortunately, little insight into the mechanism of decay is provided by the
available work. By protecting the reaction vessels from light, photolysis
seems to be ruled out as a mechanism, although, as will be discussed later,
benzidine may be sensitive to light. Microbial degradation is probably not
very important in the first degradation step of benzidine since chlorinated
water has a rate similar to unchlorinated water and the rate of benzidine dis-
appearance is extremely fast. The use of a sterilized sample would be neces-
sary to demonstrate conclusively a non-microbial mechanism.
The data are insufficient to demonstrate an air oxidation mechanism.
Such a mechanism would have been strongly implied if no benzidine disappearance
occurred in a deoxygenated lake water sample, but no such sample was run. Also,
the available information does not indicate that chlorination is an important
degradation mechanism, since the reaction rates are similar in chlorinated and
unchlorinated water. It is unfortunate that the pH of the lake water was ap-
parently not recorded, since it is quite possible that the reaction may be pH
dependent. Also, knowing the pH would have clarified whether volatilization
was an important loss mechanism, especially in aerated runs. (Under acid con-
ditions the benzidine would be present as a non-volatile salt.)
B. Photolysis Studies in Deionized, Areated Water - Experimental Design,
Results, and Evaluation
Two series of studies on benzidine photolysis in deionized water were
conducted (SOCMA, 1975 a, b). The first study CSOCMA, 1975 a) consisted of
irradiation of a 100 ppb solution of benzidine in deionized water. The 100 ppb
-------
benzidine solution was made from dilution of a 1000 ppm benzidine stock solu-
tion (added as dihydrochloride) with aerated deionized water. The solution
was placed in a 250 mfc quartz, flat bottom flask and set in a Fade-Ometer
irradiation apparatus (Atlas Electric Devices, Co., Fade-Ometer, Model 25-FR)
fitted with a xenon lamp. The xenon lamp provides light irradiation compara-
ble to sunlight in the important ultraviolet region C300 - 450 nm). Twenty-
three hours of Fade-Ometer exposure are approximately equal to 20 hours of
June sunlight, when the standardization is based on the bleaching of a stan-
dard dye cloth (Reiter, 1975). Analysis of benzidine at various irradiation
times was accomplished using a modified Chloramine-T colorimetric method. The
water sample was acidified, Chloramine-T was added, and the colored meriquinoid
complex was extracted with chloroform for quantitation. The clean-up procedure
and ethyl acetate extraction described previously were not used. Analysis was
performed on 100 mfc of solution and the detection limit was 5 ppb. The results
are presented in Table 3. For each time period, a complete flask was sacrificed
for analysis (in most cases duplicate samples were tested) in order to avoid
the effect of level changes. The reaction mixture was initially at room tem-
perature and after 2.5 and 24 hours of irradiation was at 37 - 38°C. The pH
was not reported.
The second study (SOCMA, 1975 b) used a wider variety of concentra-
tions, increased the sensitivity of the colorimetric method (limit of detec-
tion 0.1 ppb), and adjusted the pH before irradiation to 6.9 - 7.0 with sodium
hydroxide. The temperature was at 45 - 47°C which was significantly different
from the first study. The results are presented in Table 4.
Because of the experimental design of these studies, it is very dif-
ficult to determine the importance of photochemical degradation in the environment.
10
-------
Table 3. Photolysis Study of 100 ppb Benzidine in Deionized, Aerated Water
(SOCMA, 1975 a)
Time **
(hrs.)
0
0.5
0.5
1.5
1.5
2.5
2.5
4.0
4.0 .
12.0
12.0
24
Ug in Sample of 100 mfc
Test 1 Test 2
10
7.86
6.99
2.02
— *
1.63
2.22
<0.5
10.0
3.38
5.63
1.47
1.80
0.45
0.177
N.D.
0.00639
N.D.
% of initial Concentration
Test 1 Test 2
100
78.6
69.9
20.2
— *
16.3
22.2
<5
100
33.8
56.3
14.7
18.0
0.45
1.73
0.064
* Sample obviously degraded
** Samples with identical
times were duplicates
Temperature
Room Temperature
Initially, 37-38°C at
Equilibrium
11
J
-------
Table 4. Photolysis of Benzidine at Various Concentrations in Deionized, Aerated
Water Initially at 6.9 - 7.0 pH (SOCMA, 1975 b)
Time
0
0.5
1.5
4.0
12.0
24.0
27.0
28.0
29.0
48.0
53.0
56.0
72.0
77.0
80.0
1.0
0.73
0.66
0.45
0.087
< 0.002
< 0.001
-
-
-
-
-
-
-
-
Temperature
Room temperature
initially, 41°C in
1.5 hrs., remained
at 46-47°C for the
remainder of the
times
ppra Benzidine
10
8.70
8.05
6.45
4.65
2.49
-
-
1.80
0.50
0.29
-
0.018
0.018
-
Temperature
45-49°C after
first 4 hours.
100
91.95
86.93
95.61*
75.41
63.68
-
52.13
-
-
-
31.15
-
-
22.92
Temperature
Varied from 47°C
after 1.5 hrs. to
55°C.
* Irregularity in the 4 hr. value may have been caused by adsorption on the
solids which participated at the high concentrations.
12
-------
Since aerated, deionized water was used, it is possible that all the loss of
benzidine could be due to the chemical degradation noted in the previous section.
Apparently, no non-irradiated or deoxygenated water controls were prepared for
comparison. The elevated temperatures encountered in samples in the fadeometer
could have accelerated non-photolytic and photolytic breakdown. The rates of
benzidine lost in aerated lake water samples are not that different from the
losses noted in the irradiated samples. Controls consisting of an aerated,
non-irradiated sample maintained at temperatures comparable to the irradiated
samples as well as a deoxygenated, irradiated sample, would have been useful.
Varying the pH would also have facilitated interpretation, since the ultraviolet
absorption spectra is pH-dependent. The hydrogen ion concentration may also
affect the efficiency and mechanism of the photochemical process if photodegra-
dation Is taking place. Furthermore, it has been shown that photolysis in
natural waters may be substantially different than photochemical processes in
purified water (e.g., Zepp et^ ai., 1975); thus, photolysis in natural water
(perhaps lake water) would also have been useful.
C. Biological Degradation - Activated Sludge System
Biodegradation is frequently the most important process for degrading
a chemical contaminant in the environment. Very little information is available
on the biodegradability of benzidine. Lutin and coworkers (1965) studied the
biodegradation of benzidine using a Warburg apparatus, unacclimated activated
sludge C2,500 mg/Jl suspended solids), and a benzidine concentration of 500 mg/£.
They found that the oxygen uptake was less than the control, suggesting that
benzidine was not biodegraded and/or the growth of the degrading microorganisms
was inhibited by the presence of benzidine.
13
-------
The SOCMA Benzidine Task Force also has examined the biological
degradation of benzidine. In one study (SOCMA, 1975 a) benzidine was added
at a concentration of 110 mg/fc to a model activated sludge treatment plant.
The study indicated that the benzidine remained substantially unchanged in
the system effluent and had no adverse effect on the normal BOD reduction
of the system. In a later study (SOCMA, 1975 b), the activated sludge pilot
system was first acclimated to aniline at 50 tng/X and then fed 40 ppb of
benzidine (aniline remained at 50 ppm). A continuous week of feeding benzidine
at approximately 45 yg/£ had little effect on BOD-COD reduction, indicating
no toxic effects of benzidine to the microorganisms. The liquid effluent
and sludge phase were analyzed by the Chloramine-T colorimetric method in
order to provide a mass-balance of benzidine. Of the 1579.5 yg of benzidine
fed during the week run, 202.5 yg were recovered in the liquid effluent or
sludge phase. The investigators concluded that 1377 yg had been biologically
degraded or oxidized.
In the third study (SOCMA, 1975 c) a variety of conditions and effects
were studied. Warburg tests using benzidine as the test substrate were run
with both acclimated and non-acclimated sludge (2000 - 2500 mg/£ Mixed Liquor
Suspended Solids). The rate of oxygen uptake was compared to the control to
determine if the benzidine was toxic to the microorganisms. An inhibitory
effect was noted somewhere between 40 mg/£ to 80 mg/JZ, of benzidine for the
unacclimated sludge and between 60 mg/Jl to 120 mg/£ of benzidine for the ac-
climated sludge. In the first few hours the oxygen consumed was 2% of the
theoretical oxygen demand (amount of oxygen required to convert benzidine to
carbon dioxide and water) for the unacclimated sludge, and 2.8% for the ac-
climated sludge. The activated sludge pilot system described above was also
14
-------
run for six weeks after acclimation with 50 yg/£ of benzidine and no aniline.
During the six week period 14,168 yg of benzidine were fed to the system, of
which 13,506 (almost 95%) were oxidized. Adsorption to the sludge was considered.
A sterile control was run in order to determine the amount of oxidation attribu-
table to air oxidation. It was concluded that approximately 30% of the oxida-
tion could be assigned to air oxidation. Also, an unacclimated activated sludge
run was also undertaken which demonstrated approximately the same benzidine
reduction (.95%) as the acclimated test.
The above results are difficult to interpret in terms of biodegrada-
tion in nature. The microbial concentrations and communities, and nutrient
concentrations of an activated sludge treatment plant resemble secondary sewage
treatment plant conditions but are very different from natural environmental
conditions. The results from the Warburg test, which simulates activated
sludge treatment conditions, suggest some breakdown, but oxygen consumption
equivalent to 2 - 2.8% of theoretical oxygen demand is not suggestive of an
easily biodegraded compound. However, the pilot plant studies do indicate
that sizable portions of benzidine in a waste stream can be removed by a com-
bination of air oxidation and bio-oxidation. Whether similar oxidation in
natural waters will occur is still unknown.
D. Benzidine Ambient Monitoring Data
Ambient monitoring data can often provide insight into the persistence
of a chemical contaminant in the environment. Detection of a chemical usually
provides clear evidence of some resistance to degradation. However, lack of
detection is somewhat harder to interpret because there may be several reasons
for the lack of detection besides degradation (e.g., poor analytical method,
strong adsorption of the material onto soil or sediment).
15
-------
The SOCMA Task Force (SOCMA, 1975 b) conducted a field survey to
analyze water and sediment samples for benzidine in the Buffalo River Watershed
upstream and downstream from Allied Chemical Corporation's Specialty Chemical
Division Plant at Buffalo. It was believed that benzidine was being discharged
from this plant.
During July 1, 2, and 3, 1975, 21 sediment and 42 water samples were
gathered from 7 sites. Each sample was maintained in ice-water until it arrived
at the analysis laboratory (less than 20 minutes). Analysis using the Chlora-
mine-T method indicated that the concentrations of benzidine in the sediment
or water samples were below the level of detectability (~ 0.2 pg/Jl when using
1 liter samples). A similar study of water samples from the Niagara River and
near the intake for the Tonawanda Water Treatment Plant showed no detectable
benzidine.
These results suggest that benzidine may be altered in the aqueous
environment to the extent that it is no longer detectable by the colorimetric
method. However, the lack of detection could also be due to dilution below
the limit of detection. Interpretation of the results in terms of degrada-
bility would have been much clearer if the amounts of benzidine emitted into
the water systems were available as well as a calculated maximum concentration
estimated from dilution due to the flow of the rivers. It should be kept in
mind that benzidine has been detected in the Sumida River in Japan (Takemura
•et'al., 1965).
E. Comparison of SOCMA Conclusions and Conclusions Supported by Research
Data
From the studies discussed above, the SOCMA Benzidine Task Force
(SOCMA, 1975 a) has reached the conclusion that "benzidine is destroyed in
16
-------
nature by air oxidation, biological oxidation, and sunlight." In the second
submission, SOCMA (1975 b) notes in the summary that "it has been shown that
benzidine is not a persistent compound. Laboratory data have indicated that
benzidine is readily destroyed by naturally occurring processes [emphasis
added by SOCMA]." In the final submission, SOCMA (1975 c) concludes that
"benzidine is not a persistent chemical but is destroyed by bio-oxidation,
air oxidation, exposure to ultraviolet light and chlorination as practiced
in water treatment." In order to understand whether these conclusions are
justified by the experimental data, such terms as "persistence" and "destroyed"
must be defined. The term "destroyed" implies to us that the chemical is
totally broken up or degraded to products such as carbon dioxide, water, and
nitrogen oxides (usually referred to as mineralization). The term "persistence"
has had many interpretations (see Howard et al., 1975), but we prefer to define
non-persistent compounds as chemicals that are degraded in a relatively short
period of time to naturally occurring low molecular weight metabolites or that
are mineralized. However, non-persistent chemicals are not compounds that
undergo slight chemical alterations, which is all that can be measured with the
Chloramine-T method. In the detergents industry slight chemical alterations
of detergents have been referred to as "primary degradation" (Swisher, 1970).
Thus, the available information does not support the statement that
benzidine is readily "destroyed" by naturally occurring processes. Of the
three mechanisms of degradation (air oxidation, biological oxidation, or sun-
light) , only air oxidation has convincingly been shown to have such an effect.
The lack of controls in the photolysis studies and the poor simulation of nature
in the biological oxidation studies preclude any firm conclusions on these two
possible degradation mechanisms. The biological oxidation studies do indicate,
however, that degradation of benzidine is likely to occur in a secondary sewage
treatment plant.
17
-------
IV. GENERAL DISCUSSION OF ENVIRONMENTAL DEGRADATION TESTING AS APPLIED TO
AROMATIC AMINES AND CARCINOGENS
From the above discussion, it is our conclusion that the information
available on the environmental persistence or degradability of benzidine is
not sufficient to make an intelligent regulatory decision. Benzidine is a
carcinogen whose activity is thought to be due to some of its metabolites.
Without understanding the pathways and metabolites resulting from degradation
in the environment, the hazards associated with the presence of benzidine in
the environment cannot truly be assessed.
Making predictions concerning the environmental persistence of aromatic
amines as a class from the data generated for benzidine would require some
understanding of the mechanism and pathways of degradation of benzidine. Since
such information is not available in the SOCMA reports, meaningful predictions
cannot be made other than the suggestion that aromatic amines may be susceptible
to air oxidation.
From the generally negative conclusions in this report, it is obvious
that we would not recommend this benzidine testing program as a prototype for
environmental fate testing, especially for carcinogenic chemicals. This is
indeed unfortunate since the money and effort spent on the benzidine work,
with very slight modification, could have produced excellent results.
Our major criticism of the benzidine protocol is the analytical method
used. The analytical method is sensitive and specific, but it only measures
14
benzidine. By using C-labelled benzidine, which is readily available
($111/50 yCi, New England Nuclear, personal communication), the same or better
sensitivity could have been maintained, but a mass balance of benzidine and
breakdown products could have been developed. Labelling the compound also
would have facilitated isolation of the metabolites.
18
-------
The reaction conditions used with benzidine are fairly realistic considering
that benzidine's major source of contamination is from water effluents. However,
much better controls are necessary to distinguish between the mechanisms of de-
gradation. It is important to understand the mechanism of degradation in order
to determine the variability of results in nature. For example, if biodegrada-
tion is the major mechanism, the rates of degradation will vary considerably
depending upon the microbial concentration and population, carbon sources, accli-
mation, etc. However, if air oxidation is the major degradation mechanism, the
rates should be fairly constant in different microenvironments.
14
We would recommend a testing program that would include C-benzidine in
lake or river water, lake or river water with sediment, sterilized lake or
river water (with and without sediment), and distilled water. The pH of the
water should be buffered to acidic, neutral, and basic conditions (pH 5-9) to
determine the effect of the hydrogen ion concentration. Controls with and with-
out light should be run to determine the effect of laboratory light. Photolysis
studies should be run with distilled water and simulated sunlight at tempera-
tures and pH's comparable to dark samples. The effect of oxygen concentration
in irradiated and non-irradiated samples should be determined by deoxygenating
some controls (probably by bubbling nitrogen through the solution). Sediment
should be included in at least one test because benzidine has been shown to
adsorb to some clays (Furukawa and Brindley, 1973; pH-dependent adsorption,
Lahav and Raziel, 1971 a, b), and this may alter the rate and pathway of de-
gradation.
Attempts to isolate and identify metabolites will vary depending upon
14 14
where the C label resides. Traps for CO. and any volatile metabolites
19
-------
should be included so that a mass balance will be possible. Emphasis should
be placed on trying to detect some of the suspected proximate carcinogens of
benzidine. The ring dihydroxyl derivative, if it is formed, may be so water
soluble that it will be difficult to isolate. The method of Sternson (1975)
may be helpful in detecting hydroxylamine degradation products.
20
-------
V. CONCLUSION
Information developed by the SOCMA Benzidine Task Force suggests that
benzidine in natural water may be rapidly air oxidized to the extent that it
is no longer detected by a Chloramine-T colorimetric method. There is no evi-
dence, however, that the loss of color using Chloramine-T method corresponds
to a loss of carcinogenic activity. The possibility of biological oxidation
or photodegradation of benzidine taking place in aqueous systems cannot be
decided from the available information, although degradation under conditions
of secondary sewage treatment appears likely. It is suggested that further
14
studies be conducted with C-benzidine and attempts be made to determine the
degradation pathways and metabolites and to distinguish between chemical, photo-
chemical, and biological degradation.
21
-------
REFERENCES
Arcos, J.C. and Argus, M.¥. (1974), "Structure-Activity Relationships," Chap. 5
in Chemical Induction of Cancer - Structural Bases and Biological Mechanisms,
Academic Press, New York.
Butt, L.T. and Strafford, N. (1956), "Papilloma of the Bladder in the Chemical
Industry - Analytical Methods for the Determination of Benzidine and 2-
Naphthylamine, Recommended by A.B.C.M. Sub-Committee," J. Appl. Chem.,
j>, 525-39.
Furukawa, T. and Brindley, G.W. (1973), "Adsorption and Oxidation of Benzidine
and Aniline by Montmorillonite and Hectrite", Clay and Clay Minerals,
2±, 279-88.
Gadian, T. (1975), "Carcinogens in Industry, With Special Reference to Dichloro-
benzidine," Chem. Indust., 19, 821-31, (Oct. 4).
Howard, P.H., Saxena, J., Durkin, P.R., and Ou, L.T. (1975), "Review and Evalua-
tion of Available Techniques for Determining Persistence and Routes of
Degradation of Chemical Substances in the Environment," U.S. Nat. Tech.
Inform. Serv., PB 243 825-7WP, EPA-560/5-75-006.
Lahav, N. and Raziel, S. (1971 a), "Interaction Between Montmorillonite and
Benzidine in Aqueous Solutions. I. Adsorption of Benzidine on Montmorillo-
nite," Isr. J. Chem., JK6), 683-9.
Lahav, N. and Raziel, S. (1971 b), "Interaction Between Montmorillonite and
Benzidine in Aqueous Solutions. II. General Kinetic Study," Isr. J. Chem.,
2(6), 691-4.
Lutln, P.A., Cibulka, J.J., and MaIaney, G.W. (1965), "Oxidation of Selected
Carcinogenic Compounds by Activated Sludge," Purdue Univ., Eng. Bull.,
Ext. Ser. No. 118, 131-45.
Puliafico, S.J. (1975), "Evaluation of Proposed EPA Method for Benzidine and
Its Salts in Wastewaters," in SOCMA (1975 a).
Radding, S.B., Holt, B.R., Jones, J.L., Liu, D.H., Mill, T., and Hendry, D.G.
(1975), "Review of the Environmental Fate of Selected Chemicals," U.S.
Nat. Tech. Inform. Serv., PB 238-908, EPA 560/5-75-001.
Reiter, W.M. (1975), Personal communication, December 10, Allied Chemical,
Morristown, New Jersey.
Sciarini, L.J. and Meigs, J.W. (1958), "Biotransformation of Benzidine, an
Industrial Carcinogen, in the Dog," A.M.A. Arch. Ind. Health, 18, 521-30.
22
-------
SOCMA (1975 a), "First Submission of the SOCMA Benzidine Task Force to Environ-
mental Protection Agency," June 5.
SOCMA (1975 b), "Second Submission, Status Report on Benzidine Control, Benzidine
Task Force, Synthetic Organic Chemical Manufacturer's Association to the
Environmental Protection Agency," August 5.
SOCMA (1975 c), "Third Submission to the U.S. Environmental Protection Agency
by the SOCMA Benzidine Task Force," November 12.
Sternson, L.A. (1975)> "Detection of Arylhydroxylamines as Intermediates in
the Metabolic Reduction of Nitro Compounds," Experimentia, 31X3), 268-70.
Swisher, R.D. (1970), Surfactant Biodegradation, Marcell Dekker, Inc., New York.
Takemura, N., Akiama, T., and Nakajima, C. (1965), "A Survey of the Pollution
of the Sumida River, Especially on the Aromatic Amines in the Water,"
J. Air Water Pollution, JK10), 665-70.
U.S. EPA (1974), "Method for Benzidine and Its Salts in Wastewaters," in
SOCMA (L975 a).
Zepp, R.G., Wolfe, N.C., Gordon, J.A., and Baughman, G.L. (1975), "Dynamics of
2,4-D Esters in Surface Waters. Hydrolysis, Photolysis, and Vaporization,"
Environ. Sci. Technol, 9(13), 1144-50.
23
-------
TECHNICAL REPORT DATA
(Please read laaructions on the reverse before completing)
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Persistence and Degradability Testing of Benzidine
and Other Carcinogenic Compounds
5. REPORT DATE
June 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Philip H. Howard and Jitendra Saxena
8. PERFORMING ORGANIZATION REPORT NO
TR 76-571
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Center for Chemical Hazard Assessment
Syracuse Research Corporation
Merrill Lane
Syracuse, NY 13210
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
EPA 68-01-2679
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report reviews and evaluates information on the environmental degradation
of benzidine that was generated by the Synthetic Organic Chemical Manufacturer's
Association (SOCMA) Benzidine Task Force and submitted to the U.S. Environmental
Protection Agency. The experimental design, execution, and interpretation of the
studies have been reviewed and evaluated. It is concluded that the SOCMA information
is not sufficient for making intelligent regulatory decisions and a test program
using -^C-benzidine is recommended in order to generate the necessary information.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
benzidine
environmental fate testing
persistence
b.lDENTIFIEHS/OPEN ENDED TERMS
c. COSATI l-'iclcl/Group
18. DISTRIBUTION STATEMENT
Document is available to public through the
National Technical Information Service,
Springfield, Virginia 22151
19. SECURITY CLASS (This Report)
21. NO. OF PAGtS
23
20. SECURITY CLASS (Thispage)
22. PRICE
EPA Form 2220-1 (9-73)
------- |